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An ESA autosampler was used to inject 30 μL of sample during the separation run; the run time for each separation was 20 min. Isocratic HPLC separation of sugars was performed using degassed 500 mM NaOH as the eluant, utilizing a flow rate of 0.4 mL/min.
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Isocratic separation method was utilized with a flow rate of 0.7 ml/min and column temperature set to 25 °C.
An aqueous eluent (0.1 M Na2SO4/1 v/v % acetic acid) was utilized at a flow rate of 0.4 mL/min.
Samples were infused utilizing a Harvard Instruments Syringe Infusion Pump 22 at a flow rate of 10 ml/min.
Ultrapure-grade helium (99.9999%, Sabalan Co., Iran) was utilized as the carrier gas at a flow rate of 34 mL/min.
The protein sample was delivered by nanoelectrospray ionization with a custom-pulled tip at a flow rate of 20 50 nL/min, utilizing a capillary voltage of 1.2 1.4 kV, and a drying-gas temperature of 30 °C at a flow rate of 2.5 L/min.
For the gradient elution, a binary mobile phase of an aqueous solution of water (A) and methanol (B) at a flow rate of 0.5 mL/min was utilized.
An aqueous solution of a chelate compound of Gd was impulsively injected into the dialysate flow path at a flow rate of 500 cm3 /m, which is that utilized in actual dialysis.
A mobile phase consisting of 20 mM KH2PO4 (pH 2.9) operating at a flow rate of 0.7ml/min at ambient temperature was utilized.
Samples were separated using an Äkta Purifier (GE Healthcare) medium pressure liquid chromatography system equipped with a Mono S PC, 1.6 mm × 50 mm column (GE Healthcare) at a flow rate of 0.1 ml/min (green stages), or a BioCAD workstation (Applied Biosystems) utilizing a 100 × 4.6 mm polysulfoethyl aspartamide column (PolyLC Inc, Columbia, MD, USA) at a flow rate of 0.5 ml/min (ripe stages).
Typically, a 30 min linear gradient (from 100% MPA to 100% MPB) was utilized to elute proteins followed by MPB isocratically for 5 min to ensure elution, at a flow rate of 1 mL/min.
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